1. Field of the Invention
Quantitative and qualitative determination of amino acids in protein hydrolysates, body fluids and other physiological samples, is normally accomplished by employing ion-exchange chromatography using a cationic or anionic exchanger column. The analyte is placed on the column at a pH of about 3, at which pH the individual amino acids are positively charged. The column is then developed by gradually increasing the pH and ionic strength of the buffers with which the column is eluted.
As the column is developed, the acidic amino acids are removed from the resin first, followed by the neutral amino acids, and finally by the basic amino acids. As the amino acids are eluted from the column, they are reacted with ninhydrin or a fluorescer and the resultant color or fluorescence intensity is measured spectrophotometrically. Several automatic amino acid analyzers are commercially available which automatically separate the amino acids on an ion exchange column, contact the separated amino acids with ninhydrin, heat the mixture to develop the color, record the color intensity, at selected wavelengths, usually 440 nm and 570 nm, and plot the intensity on a graph. The identity of each amino acid is established on the basis of its migration characteristics and thus its position on the chromatogram; and the amount of amino acid is estimated quantitatively on the basis of the area under each peak of the curve as compared to that of a standard mixture.
In the development of a ninhydrin reagent for use in the automatic amino acid analyzers, there are several factors that need to be considered. Ninhydrin is not very soluble in aqueous media and solutions of ninhydrin are very unstable under normal experimental condition being highly susceptible to oxidation. For practical purposes the reagent must include a water miscible, preferably colorless organic solvent which solubilizes the hydrindantin and ninhydrin, as well as the blue compound (diketohydrindylidenediketohydrindamine) formed by the reaction of ninhydrin with amino acids.
The organic solvent used should be nontoxic to avoid expensive ventilation. Also, the solvent should not react with any of the solution components or products. In addition, during use of the reagent, precipitation of any of the components must be avoided or lines in the analyzer will become clogged. The available ninhydrins all have one or more shortcomings.
It would therefore be desirable to develop a ninhydrin reagent which would be stable to oxidation, which would exhibit pH and thermal stability, and a long shelf life. During analysis the reagent should provide flat base lines, sharp resolution, and good reproducibility of results.
2. Description of the Prior Art
Moore (J. Biol. Chem. 243, 6281 (1968)) teaches the use of aqueous dimethyl sulfoxide as a solvent for the ninhydrin reagent. Takahashi (J. Biochem. 83, 57 (1978)) teaches the use of sodium borohydride, in the preparation of a ninhydrin reagent, to prevent the formation of precipitates in the flow lines of the analyzer. Moore and Stein (J. Biol. Chem. 211, 907 (1954)) used methyl cellosolve (the monomethyl ether of ethylene glycol) as the organic solvent in ninhydrin reagents. The Merck Index, 8th Ed. lists sulfolane as an organic liquid which at 30.degree. C. is miscible with water and is used as a selective solvent for liquid-vapor extractions.